This proposal is designed to elucidate the stepwise mechanisms of formation of voltage-gated K+ channels (Kv) and the specific protein-protein interactions that underlie the acquisition of secondary, tertiary, and quaternary Kv channel protein structure. Kvl.3, a Kv channel important in the physiology of T-lymphocytes, will be the primary channel studied in the proposed research. Determinants of protein folding are key to understanding not only biogenesis, but also trafficking mechanisms because ultimately the retention/export and degradation signals are modulated by how the protein is folded/packed. Defects in any biogenic or trafficking steps will result in altered expression of Kv channels at the cell surface. Indeed, these defects lead to pathology and can be lethal. The link between folding and trafficking defects is generalizable to many non-channel disorders and underscores the need to elucidate basic principles of folding and oligomerization in Kv biogenesis. This is the goal of our proposal. The overall goal is divided into six specific aims. The first four aims are devoted to identifying key folding and oligomerization interactions in the T1 recognition domain and the pore region of Kvl.3 during biogenesis. The fifth aim is devoted to assessing when and in which compartment secondary Kv conformations are achieved. The sixth aim is devoted to identifying protein-protein, protein-lipid, and protein-aqueous interfaces during assembly. The strategies proposed in this application, namely pegylation, mass-tagging, and crosslinking, will enable electrically silent residues to be probed for their role in biogenesis. Moreover, translocation intermediates of Kv 1.3 will be made and studied. The experiments will employ a range of techniques, including biochemical and electrophysiological assays in Xenopus oocytes and microsomal membranes of the endoplasmic reticulum.